Rubiflavin and a process for making same using streptomyces griseus



RUBIFLAVIN AND SAME USING ST Edward Pansy, Jamesb and Robert S. Robi bymesne assign A PROCESS FOR MAKING OM YCES GRISEUS dorjan Aszalos,Kenson, North Brunswick, ments, to E. R. Squibb a corporation of FiledAug. 25, 1964, Ser. 2 Claims. (Cl. 16

(ATCC 15569) produces the antiflavin by culturin ut 23 to 30Streptomyces griseus cterial antibiotic rubi t-ure in the range of abo25 C., under submerge nutrient medium containing g at a tempera- C.,preferably about bic conditions in an aqueous an .assimilable,fermentable ose and glucose, etc.

asparagine, casein hydrolyliquor, beef extract, yeast carried out forabout one At the end of this mount of rubiflavin has been Rubiflavin maybe obtain fermentation broth with about 7.6, then extractin with a waterimmiscible ylacetate, benzene or th solvent extracts are combinevaporated. The oily resid volumes of ether or forms is separated a Dryhydrogen chloride is i supernatant and the h separated by centrifugiether and drying, flavin free base solution and then such as those meered from the organic phase as be further purified, e.g., by countsolvent system using ethyl acetate buffer system at about pH 7.5 an

perature, e.g., about 3 to 10 C. in a Craig app fication, the stepsrepeated. The extracts are extra other water immiscible solvent de andthe solvent evaporated. The der is dissolved in ether, HCl gas chloridesalt is centrifuged. Th with dilute aqueou ed by first filtering thecrude out adjusting the pH, which is g both the filtrate and filter cakesolvent such as chlo preferably the first. ed, dried and the solvent isue is suspended in 15 to 20 The precipitate which ntroduced into theether ydrochloride salt which forms is After washing with converted intorubisodium carbonate immiscible solvent The rubiflavin is recova redpowder and may rcurrent extraction.

ng or the like.

the hydrochloride is with dilute aqueous treated with a water ntionedabove.

d a temperature below about room tem erably about 5 For final puriaratus is preferably used. described before may be cted with chloroformor scribed previously, dried residual brick red powis added and thehydrois is then reconverted to s sodium carbonate solutracted again withwater he solvent evaporated to the free base The free rubi-flavin is eximmiscible solvent, dried and t leave the pure product.

Rubiflavin is soluble in a acetone, dimethylsulfoxide,

cidic water, methanol, ethanol, ethyl acetate, chloroform, ben- UnitedStates Patent 3,314,853 Patented Apr. 18, 1967 ride, phosphate, oxalate,maleate, picrate, p-nitrobenzenesulfonate, naphthylsulfonate orreineckate.

The following example is illustrative of the invention.

EXAMPLE The microorganism Streptomyces griseus (ATCC 15569) is isolatedfrom a soil sample by conventional streaking methods.

a thin gruel, ml. of water and steri-lzed at 121 C. and 15 pounds steampressure for 20 minutes.

sterilized broth in a 250 ml. flask. The broth has the followingcomposition (on a weight basis):

Soybean meal, percent 1.5 Dehydrated mashed potato, percent 1.5 Glucose,percent 5.0 CoCl .2H O, percent 0.0005 CaCO percent 1.0 Distilled Water,ml. to 1000 Autoclave at 121 30 minutes.

After 48 hours incubation at 25 the contents of the flask are the abovesterile medium in a 4000 ml. flask. After 48 hour incubation on a rotaryshaker at 25 C, the entire contents of the flask are used to inoculate a100 gallon germinator tank containing gallons of a sterile nutrientmedium having the following composition (by weight) C. and 15 poundssteam pressure for C. on a rotary shaker, transformed to 1000 ml. of

, Percent Soybean meal 3.0 Glucose 5.0 CaCO 0.7 Corn steep liquor 0.25Defoamer (prime burning oil) 7 0.05

Growth is continued for an additional 48 hours at 25 C., with an airrate of 1.0 ft./min. superficial air velocity and agitation equivalentto 0.2 H.P./ gal. At this time approximately 40 gallons of thegerminator inoculum is used to inoculate 800 gallons of the followingmedium contained in a 1300 gallon fermentor:

Percent Soybean meal 3.0 Glucose 5.0 C3003 Corn steep liquor 0.25Defoamer (prime burning oil) 0.05

The fermentation is continued for hours at 25 C. The air rate is 15ft./min. superficial air velocity and agitation equivalent to 0.2 H.P./100 gal. is used.

of the fermentation period, the 800 gallon fermentation broth isfiltered. The filtration broth and the filter cake are extractedseparately with chloroform. Chloroform in the amount of /3 of the volumeof the filtered broth is used for extracting the filtrate and thischloroform solution is then used to extract the filter cake. Extractionis carried out at harvest pH (approximately 7.6) and at roomtemperature.

The chloroform extract is dried with sodium sulfate overnight, then thesodium sulfate is filtered off and the chloroform is evaporated. Theoily residue is suspended in to volumes of dry ether. The precipitatewhich forms is centrifuged off and discarded. Dry HCl gas is introducedinto the ether supernatant and the solid hydrochloride salt ofrubiflavin which forms is centrifuged, washed with dry ether and driedin vacuo. A 4% aqueous sodium carbonate solution and chloroform arequickly shaken with the salt (the amount of each solvent added is 50times the weight of the hydrochloride).

After separation, the water phase is adjusted to pH 10 and extractedonce more with chloroform. All chloroform extracts are combined, driedwith sodium sulfate, fiiltered and evaporated. The dry, red powderobtained by evaporation yields about 150 grams of product.

Two grams of the crude product thus obtained are dissolved in 20/20 ml.of the following system: 0.1 M aqueous phosphate buffer, pH 7.5, andethyl acetate equilibrated at 5 C. Ten ml. of each of the upper andlower phases are put into the first two tubes of a Craig extractioninstrument and 39 transfers are made at 5 C. Both upper and lower phasesfrom tubes Nos. 9 to 19 are collected. The combined upper organic phasesare collected and the lower aqueous phases are shaken twice withchloroform. The upper phases and chloroform extracts are are combinedand dried with sodium sulfate. The solvent is evaporated and theresidual brick red powder is collected.

The red product is further purified by dissolving in about 500 ml. ofdry ether. Dry 1101 gas is introduced and the precipitated hydrochloridesalt is collected by centrifuging. Then it is washed with ether anddried. This salt is added to 500 ml. of 4% sodium carbonate solution and500 ml. of chloroform, quickly shaken and reextracted with an equalvolume of chloroform. The chloroform phases are dried and the solventevaporated to obtain the pure product rubiflavin.

Rubifiavin has the following average elemental analysis: C, 68.50; H,7.50; N, 3.45%, O (by difference), 20.55. No halogen or sulfur ispresent. The molecular formula is C H NO and an equivalent weight of 400has been assigned. Analysis by ultracentrif-ugation and from thesedimentation and diffusion constants, a molecular weight of 412 iscalculated which confirms the above. It is an amorphous red powder whichhas no well defined melting point but melts in the range of 149 to 15 7C.

Rubuflavin has absorption bands (min.) in the infrared spectrum at 2.97,3.45, 6.06, 6.31, 6.83, 6.95, 7.02, 7.27, 7.95, 8.12, 9.12-9.25 microns(KBr pellets) as shown in the accompanying figure. It absorbs in theultraviolet and visible range as follows: maximum at 244 m and 428 mwith shoulders at 265 m 395 mi and 446 mu (absolute ethanol).

The antibiotic is soluble in acidic water, methanol, ethanol, butanol,ace-tone, dimethylsulfoxide, ethyl acetate, chloroform, benzene andether. 'It is very slightly soluble in distilled water and slightlysoluble in hexane. It is unstable in water and alcohols but is stable inethyl acetate, chloroform, benzene and ether. It turns yellow in acidicWater and violet in alkaline water; the color change is reversible.Rubiflavin gives a positive permanganate test, reacting very rapidly,and a negative reaction with ferric chloride,2,4-dinitrophenylhydrazine, Tollers reagent and ninhydrin. The HinsburgMethod for distinguishing amines shows the compound to have a secondaryamine. The compound has one N-methyl group per Upon completion 400equivalent weight. it cannot be reduced at atmospheric pressure withplatinum or palladium catalyst in ethyl acetate.

Rubiflavin is acetylated by adding 1 ml. of acetic anhydride to asolution of 30 mg. of the compound in 1 ml. of pyridine and heating at50 C. for 24 hours. All volatiles are evaporated and the yellow solidresidue is dried in vacuo at 25 C. The acetyl number is 16%. Theacetylated compound has absorption bands (min) in the infrared region at3.42, 5.64, 5.74, 5.95, 6.04, 6.27, 6.38, 6.82, 6.95, 7.30, 7.54, 7.65,8.10, 8.43, 9.22 and 9.55 microns (KBr pellets).

Rubiflavin forms acid salts with inorganic and organic acids, e.g.,hydroha'lides such as hydrochloride, hydrobromide, etc., sulfate,phosphate, oxalate, malonate, etc.

R-ubiflavin shows the following in vitro spectrum against gram negativeand gram positive organisms:

Table I.In vitro spectrum Minimum inhibitory Organism: conc. (meg/ml.)

Staphylococcus aureus 209 P 1.4 Staphylococcus aureus Cahill 3.9Bacillus subtilis Bacillus cereus 1.8 Streptococcus agalactiae 0.9Proteus vulgaris 37.5 Pseudornonas acruginosa 4.7 Salmonellaschottmuelleri 4.7 Salmonella typhimurium 9.4 Candida albicans 9.4 Trichophyton mentagrophytes 15. 6 Fusarium bulbigenum 25.0

Rubifiavin is distinguishable from other antibiotics. It can bedistinguished from pluramycin by Craig analysis. After 50 transfers inan ethyl aCetatepH 6.55 phosphate buffer system, pluramycin can berecovered from tubes 30 to 49, while rubiflavin remains at the origin.Pluramycin, after 30 transfers in an ethyl acetate-pH 5.3 phosphatebuffer Craig system, has an activity peak at tube 4; rubiflavin does notmove at all. No absorption of pluramycin is reported in the visiblerange but absorbs at 219 m while rubiflavin has no absorption at thatpoint.

Rubiflavin may be distingiushed from streptovaricin by paperchromatography. -In the system methanol-benzenewater (1: 1:2) and usingSchleicher Schule No. 589 paper, rubiflavin does not move while alltypes of streptovaricin do move. Moreover, streptovaricin is nearlyinsoluble in ether and hexane while rubiflavin is soluble in bothsolvents.

Anthracidin A and B are distinguishable in that they are stable at pH 5through 9 while rubiflavin is not. The infrared and ultraviolet spectraof the anthracidins and rubiflavin differ from each other.

Rubiflavin has antibacterial activity, e.g., against both gram positiveand gram negative organisms such as Staphylococcus aureus, Proteusvulgaris, Pseudomonas aeruginosa, Salmonella schoztmuelleri andTrichophyton mentagrophytes being useful primarily as the antibacterialagent in disinfectants or sterilizing solutions or as a preservative inplastics, paints or fabrics. It also shows cytotoxic activity bysuppressing the growth .of Carcinoma CA-755 in mice and extending thelife of mice affected with Leukemia L-l2l0 as well as suppressing thegrowth of KB kidney cells in tissue culture at dilutions ranging from1:170,000 to 1:22-0g000.

What is claimed is:

1. An antibiotic selected from the group consisting of rubiflavin andsalts thereof, said rubiflavin being a red amorphous material having thefollowing average elemental analysis: C, 68.5; H, 7.5; N, 3.45; O,20.55; a molecular formula of C gH NO a molecular weight about 400;melting in the range of about 149 to 157 265, 395 and 446 m, (inethanol); and an infrared absorp- 5 tion spectrum as shown in thedrawing.

2. A process for producing the antibiotic of claim 1 which comprisesculturing Streptomyces griseus ATCC 15569 under aerobic conditions in anaqueous nutrient medium comprising an assimilable, fermentablecarbohydrate and assimilable organic nitrogen source for about UNITEDSTATES PATENTS 2,798,834 7/1957 Hobson 167-65 2,885,326 5/1959 Churchill195-80 3,092,550 6/1963 Gaeumann et a1 167-65 ALBERT T. MEYERS, PrimaryExaminer. DAREN M. STEPHENS, Assistant Examiner.

1. AN ANTOBIOTIC SELECTED FROM THE GROUP CONSISTING OF RUBIFLAVIN ANDSALTS THEREOF, SAID RUBIFLAVIN BEING A RED AMORPHOUS MATERIAL HAIVNG THEFOLLOWING AVERAGE ELEMENTAL ANALYSIS: C, 68.5; H, 7.5; N, 3.45; O,20.55; A MOLECULAR FROMULA OF C23H29NO5; A MOLECULAR WEIGHT ABOUT 400;MELTING IN THE RANE OF ABOUT 149* TO 157* C.; SOLUBLE IN METHANOL,ETHANOL, BUTANOL, ACETONE, DIMETHYLSULFOXIDE, ETHYL ACETATE, CHLOROFORM,BENZENE AND ETHER; ABSORPTION BANDS IN THE ULTRAVIOLET AND VISIBLESPECTRA AS FOLLOWS; MAXIMA AT 244 AND 428MU AND SHOULDERS AT 265, 395AND 446 MU (IN ETHANOL); AND AN INFRARED ABSORPTION SPECTRUM AS SHOWN INTHE DRAWING.
 2. A PROCESS FOR PRODUCING THE ANTIBIOTIC OF CLAIM 1 WHICHCOMPRISES CULTURING STREPTOMYCES GRISEUS ATCC 15569 UNDER AEROBICCONDITIONS IN AN AQUEOUOS NUTRIETN MEDIUM COMPRISING AN ASSIMILABLE,FERMENTABLE CARBOHYDRATE AND ASSIMILABLE ORGANIC NITRROGEN SOURCE FORABOUT THREE TO SEVEN DAYS AND RECOVERING RUBIFLAVIN FROM THE MEDIUM.